Objective To further study the mechanism of LHRH pulsatility in non-human primates. ABSTRACT:Previously, we have hypothesized that the local concentration of LHRH in the stalk-median eminence (S-ME) is crucial for the pulsatility of LHRH release. This is based on the finding that infusion of an LHRH agonist at 10 nM into the S-ME in ovariectomized rhesus monkeys altered the pulsatility of LHRH release. To further study the mechanism of LHRH pulsatility in non-human primates, the present study examines the role of EP24.15, an enzyme which cleaves LHRH at the Tyr5-Gly6 bond. First, the presence of EP24.15 was confirmed in the monkey hypothalamus by immunocytochemistry and Western immunoblotting. Second, using push-pull perfusion, a specific antibody to EP24.15 was infused into the S-ME for 10 min in peripubertal and ovariectomized adult monkeys, while perfusates were collected for LHRH measurement by RIA. The infusion of the EP24.15 antibody (1:500 dilution) promptly increased LHRH levels by 3-25 fold for 10-20 min (p<0.01), whereas infusion of normal rabbit serum (same dilution) did not cause any significant effects. The LHRH1-9 fragment did not cross react with the antiserum, suggesting that LHRH1-10 was the only peptide moiety measured in the assay. Third, an additional study in ovariectomized monkeys with push-pull perfusion indicated that infusion of recombinant EP24.15 at 0.05-0.5 nM suppressed LHRH release. These results indicate that removal of endogenous EP24.15 by immunoneutralization increases LHRH levels, whereas application of EP24.15 suppresses LHRH release. Furthermore, EP24.15 appears to be active in degrading LHRH1-10 in the monkey hypothalamus and the local concentration of LHRH1-10 in the S-ME may be partly controlled by EP24.15. Thus, autoregulation of LHRH1-10 controlled by EP24.15 seems to be important for the pulsatility of LHRH release. Keywords LHRH release, endopeptidase, LHRH1-5, autoregulation